Growing keratinocytes - (Jun/01/2011 )

I just started my masters and working on keratinocyte fibroblast co-culture. I have just started growing commercial keratinocytes which were in liquid nitrogen. I had two different passage numbers 23 and 28. I have not grown this cells before. So i'm a bit disturbed because they've been growing for three weeks and are not confluent. I grow them in Kert serum free media (K-SFM), 35ng/ml EGF, 0.05mg/ml BPE and 0.5% pen/strep/amp in proportion 49.4mls /1.1ul/250ul/500ul. At moment part of the flask is confluent and another part is still very scanty and I'm contemplating whether to trysinise and put back in another flaskor just keep changing the media. Initially, I was changing the media twice a week and resolved in changing it after evry two days. sincerely i need to know how to keep my cells happy and get them growing. I need an experts advice before I lose my cells. thank you.

-sandram-

Growing keratinocytes in serum-free media is not so easy. Is there a reason for not using serum? If you want more information on growing keratinocytes, call Genzyme Biosurgery in Cambridge, MA, USA.

-Don Boyce-

Don Boyce on Fri Jun 3 18:53:12 2011 said:

Growing keratinocytes in serum-free media is not so easy. Is there a reason for not using serum? If you want more information on growing keratinocytes, call Genzyme Biosurgery in Cambridge, MA, USA.

Dear Boyce,
thank you for that information. Actually, previous studies have used that and that was like the suggested media.I just lost all the cells. Please can you send me the email address.
Thank you,

-sandram-

sandram on Mon Jun 6 09:20:08 2011 said:

Don Boyce on Fri Jun 3 18:53:12 2011 said:

Growing keratinocytes in serum-free media is not so easy. Is there a reason for not using serum? If you want more information on growing keratinocytes, call Genzyme Biosurgery in Cambridge, MA, USA.

Dear Boyce,
thank you for that information. Actually, previous studies have used that and that was like the suggested media.I just lost all the cells. Please can you send me the email address.
Thank you,

serum induces keratinocyte differentiation.

-vecttoraz-

Hi i remember growing the cells using a growth supplement, it might help you. Try contacting a supplier of supplements, we currently use Jisslab's range of OzyCells supplements... their contact details are jisslab@aol.com